Abasic site endonuclease assay

ABSTRACT

The present invention provides a novel method for detection and/or genotyping of nucleic acids that utilizes the specificity of an AP endonuclease. In addition, the present invention provides a novel method for nucleic acid amplification.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of provisional application Ser. No.60/405,642, filed on Aug. 21, 2002, the disclosure of which is herebyincorporated herein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK.

NOT APPLICABLE

BACKGROUND OF THE INVENTION

Apurinic/Apyrimidinic (AP), or abasic, sites arise spontaneously in DNAwith a calculated rate up to 10,000 bases per human cell per day. APsites are cytotoxic and mutagenic and need to be repaired quickly inorder to maintain the functional and genetic integrity of the genome.One of the major sources of AP sites is inherent instability of theglycosylic bond, found predominantly in purines. Abasic sites can alsoarise either by the actions of reactive oxygen species, or by enzymaticexcision of damaged bases via the cleavage of the N-glycosyl bondcatalyzed by a DNA glycosylase (See: Prokaryotic Base Excision Repair,Wilson III, D. M., Engelward, B. P. and Samson, L. (1998) pp.29-64;from: DNA Damage and Repair, V.1: DNA Repair in Prokaryotes and LowerEukaryotes, Edited by: J. A. Nickoloff and M. F. Hoekstra, humana PressInc., Totowa, N.J.).

AP sites in double-stranded DNA are recognized by a class of enzymestermed Class II AP endonucleases that cleave the phosphodiester backboneon the 5′ side of the AP site via a hydrolytic mechanism, therebyproviding a free 3′-OH group that serves as a substrate for DNApolymerases to initiate Base Excision Repair (BER). The Endonuclease IVfrom Escherichia coli is one example of a Class II AP endonuclease. See:Regulation of Endonuclease IV as Part of an Oxidative Stress Response inEscherichia coli, Weiss B. (1998) pp.85-96; from: DNA Damage and Repair,V.1: DNA Repair in Prokaryotes and Lower Eukaryotes, Edited by: J. A.Nickoloff and M. F. Hoekstra, humana Press Inc., Totowa, N.J.

A number of DNA glycosylases that are called Class I AP endonucleasesexhibit AP site-cleavage activity as part of their mechanism of action.However, these enzymes act as β-elimination catalysts, cleaving thephosphodiester backbone 3′ to the AP site, resulting in atypical3′-termini, such as 3′-phosphoglycolate and 3′-phosphate. These atypicaltermini block the 3′-OH group that serve as a substrate for polymerasesand are subject to subsequent repair by the Class II AP endonucleasesthat cleave the blocks and initiate the BER. See: Prokaryotic BaseExcision Repair, Wilson III, D. M., Engelward, B. P. and Samson, L.(1998) pp.29-64; from: DNA Damage and Repair, V.1: DNA Repair inProkaryotes and Lower Eukaryotes, Edited by: J. A. Nickoloff and M. F.Hoekstra, humana Press Inc., Totowa, N.J.; and Abasic Site Repair inHigher Eukaryotes, Strauss, P. R. and O'Regan, N. E. (2001) pp.43-86;from: DNA Damage and Repair, V.3: Advances from Phage to Human, Editedby: J. A. Nickoloff and M. F. Hoekstra, humana Press Inc., Totowa, N.J.

Polynucleotide identification assays that are based on a selectivecleavage of a probe hybridized to a target nucleic acid have beendisclosed by others. For example, U.S. Pat. Nos. 4,876,187; 5,011,769;5,660,988; 5,731,146; 5,747,255 and 6,274,316 disclose nucleic acidprobes having a scissile linkage incorporated as part of the nucleicacid backbone and in the middle of the nucleic acid probe. U.S. Pat. No.5,403,711 also discloses a similarly designed DNA-RNA-DNA probe, whereinthe embedded RNA sequence is a substrate for RNase H when duplexed.Hybridized probes with an incorporated cleavable linkage within themiddle of the probe have a diminished duplex stability after theenzymatic cleavage. Their cleavable sites also are not exquisitelyspecific.

U.S. Pat. Nos. 5,516,663 and 5,792,607 disclose using endonuclease IV toremove an abasic site incorporated as a blocking agent on the 3′ end ofan oligonucleotide to improve specificity and sensitivity of the ligasechain reaction (LCR) or polymerase chain reaction (PCR) amplification.

U.S. Pat. Nos. 5,656,430; 5,763,178; 6,340,566 disclose methods fordetecting point mutations by using an endonuclease to cleave the nucleicacid backbone in the middle of the oligonucleotide at the point ofmutation. In methods that identify a mismatch by enzymatic cleavage of anucleic acid backbone, the presence, rather than the absence, of amismatch stimulates the cleavage of the probe phosphodiester backbone.

U.S. Pat. No.6,309,838 discloses using labeled nucleotide excisionrepair enzymes to detect bound enzyme to DNA sequence impairments.

European Patent EP 1 071 811 B1 discloses a method of DNA synthesis froma 3′-OH generated by cleavage with a DNA glycosylase, but this methodrequires the steps of introducing a modified base and excising themodified base with a glycosylase followed with a treatment by APendonuclease before carrying out the extension.

What is needed in the art is an assay which combines the advantages oftarget nucleic acid cycling, retained binding stability of the probe, anexquisitely specific cleavage site, the possibility for essentiallyinstantaneous and highly sensitive reporter detection and the ability todirectly combine detection with amplification procedures. Accordingly,there remains a need for compositions and methods that enable efficientdetection of target nucleic acids with exquisite specificity. Thepresent invention fulfills this need and others.

BRIEF SUMMARY OF THE INVENTION

Provided is an AP site probe comprised of an oligonucleotide NA thathybridizes to a target nucleic acid, and a functional tail R comprisinga detectable reporter group and an AP endonuclease cleavage siteattached through a phosphodiester bond of a phosphate group to the 3′terminal nucleotide of the NA, wherein the reporter group is notdetected when the functional, chemical tail R is attached to the NA.

The AP site probes find use in methods and assays for detecting a targetnucleic acid of interest in a sample. The methods involve contacting thesample with at least one AP site probe and an AP endonuclease, underreaction conditions sufficient to allow the at least one AP site probeto hybridize to the target nucleic acid and form a reaction mixture,incubating the reaction mixture under reaction conditions that allow theAP endonuclease to cleave the phosphodiester bond attaching thefunctional tail R to the 3′ terminal nucleotide of the NA, and detectingthe reporter group on the cleaved functional tail R. The methods areexquisitely sensitive to the detection of single base pair mismatchesbetween a probe NA component and a target nucleic acid because the APendonuclease preferentially cleaves the phosphodiester bond attachingthe tail R to the NA when the NA is hybridized with a fullycomplementary nucleic acid sequence in comparison to cleaving afunctional tail attached to a NA that is unhybridized or hybridized to anon-complementary nucleic acid.

The invention further provides primers with internal APendonuclease-cleavable sites (pL), the primers having a sequencestructure (NA₁-L)_(m)-NA₂, wherein NA₁ and NA₂ are nucleic acidsequences complementary to the target nucleic acid, L is an APendonuclease-cleavable linker, and m is from 0 to 100, where at leastone of the forward primer and the reverse primer comprises an APendonuclease-cleavable linker, L. The primers find use in methods foramplifying a target nucleic acid sequence of interest in a sample, themethods involving contacting a sample with at least one forward and atleast one reverse primer having internal AP endonuclease-cleavablesites, an AP endonuclease, and a nucleic acid polymerase, underconditions sufficient to allow the forward and reverse primers tohybridize to the target nucleic acid and form a reaction mixture, andincubating the reaction mixture under reaction conditions thatsimultaneously allow the AP endonuclease to cleave at a linker site L,thereby generating a free 3′-OH, and the polymerase to extend theprimers in a template-specific manner.

The invention further contemplates kits containing reagents, includingat least one AP site probe, for carrying out the described methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of two types of DNA lesions cleaved byAP endonucleases. “a^(x)” represents an abasic site, 2′-deoxyribose. “s”represents products of the 2′-deoxyribose (abasic site). Spontaneous orenzymatic degradation leads to cleavage of the phosphodiester bondbetween the 3′-hydroxyl group of the ribose (abasic site) and thenearest nucleotide of the DNA strand. Lesion 1 is a typical AP, orabasic, site generated by loss of a nuclear base. Lesion 2 is anatypical abasic site that appears as a result of inherent instability ofthe deoxyribose in Lesion 1 or its cleavage by a Class I APendonuclease.

FIG. 2 illustrates a schematic diagram of the probe-enhancer-targetnucleic acid complex that is recognized and cleaved by an APendonuclease at the linkage shown by arrow. The presence of anenhancer-target duplex is not required for tail-cleavage. However, anenhancer-target duplex bound downstream from the probe usually improvesthe kinetics of the tail-cleavage reaction.

FIG. 3 illustrates a schematic diagram of a solid support base assaythat incorporates both tail cleavage and probe extension reactions fortarget nucleic acid detection. Probe having a cleavable tail isimmobilized on a solid support. The target nucleic acid, enhancer andprobe comprise a substrate complex that facilitates endonucleasecleavage of the -p-R tail, resulting in a 3′-OH group on the probe thatcan be extended by a nucleotide polymerase. Nucleotide 5′-triphosphates(NTPs) are labeled with a specific label or marker. In the exampleshown, polymerization is terminated after incorporation of a labelednucleotide. After the removal of the excess labeled NTPs by washing, thelabeled probe is bound to the solid support and presence of the targetnucleic acid duplexed with the probe can be detected. In addition to thetarget nucleotide detection, this approach allows the determination ofthe sequence of the target nucleotide that is 3′ from the duplexedregion. In such cases, every NTP needs to be labeled by a specificmarker. “R” represents a functional chemical tail.

FIG. 4 illustrates a schematic diagram of an assay that incorporatesboth the tail cleavage and probe extension reactions for target nucleicacid detection. “D1” is a fluorescent dye and “DX” is a fluorescent dyeother than D1. Both dyes are selected to support a FluorescenceResonance Energy Transfer (“FRET”) effect so emission of D1 overlapswith the absorbance of the DX. FRET is used to distinguish two or moredyes within the same molecule or complex from two or more dyes attachedto different molecules. The reaction mixture is irradiated at awavelength that is within the absorbance of D1 but not DX; fluorescenceof the reaction mixture is measured within the range of emission of theDX dye. DX dye is detected in the mixture only when this moiety isincorporated into the probe sequence. DX could be one or more dyes. Forexample, every NTP can be labeled with a particular dye. For an optimalFRET effect, D1 is preferentially conjugated close to the 3′ end of theprobe. The conjugation must not block either the tail-cleavage site orthe probe extension reactions.

FIG. 5 illustrates a schematic of exemplified linkage strategies to link5′-end of a probe and 3′-end of an enhancer either covalently (A) ornon-covalently (B).

FIG. 6 illustrates a schematic diagram that shows the initial stages ofa target nucleic acid amplification by a method of the presentinvention. At stage A, a primer that has multiple, endonucleasecleavable linker sites (“pL”) incorporated randomly through its sequencebinds to the target nucleic acid strand. At stage B, polymeraseextension originates from a 3′ end of a primer to provide a duplex. Atstage C, an endonuclease cleaves the pL linker sites, providingavailable 3′-hydroxy groups for polymerase extension. At stage D, asubsequent extension reaction originating from an cleavable linker sitedisplaces a previously synthesized strand. This method allows multiplecopies of a complementary target nucleic acid strand to be synthesizedfrom one target nucleic acid strand.

FIG. 7 illustrates exemplified fluorescein flurophores and linkers thatcan be incorporated in the functional tail R

FIG. 8 illustrates exemplified cleavable quenchers and linkers that canbe incorporated in the functional tail R. Quencher molecules withoutcleavable linkers can be incorporated in the middle or at the 5′ end ofan AP site probe. Structure 15 is an example of incorporation of apreferred quencher to the 5′-end of AP site probe.

FIG. 9 illustrates the preferred hydroxyprolinol linker and compares itsstructure to a natural abasic site.

FIG. 10 illustrates a hairpin structure (SEQ ID NO:1) simulating aprobe-target nucleic acid-enhancer complex as a model substrate for anAP endonuclease. Cleavage of this substrate in reaction with E. coliendonuclease IV is also shown on FIG. 10. The reaction was monitored asfluorescence vs. time in 5 mM MgCl₂, 20 mM Tris-HCl (pH8.5). Experimentwas performed on ABI PRISM™ 7700 Sequence Detector at 60° C. with thehairpin substrate concentration of 150 nM and the enzyme concentrationof 0.0004 U/μL. Structure of the tail used in this example is shown inFIG. 7, structure #2.

FIG. 11 illustrates the effect of including an enhancer molecule in anAP Endonuclease tail-cleavage assay. The assay is described in Example1, infra.

FIG. 12 illustrates the efficiency of the tail-cleavage reaction as afunction of probe hybridization properties, i.e., melting temperature(T_(m)). The assay is described in Example 2, infra.

FIG. 13 illustrates substrate specificity of the Endonuclease IV enzymein the presence of varying gap sizes between the AP site probe and anenhancer molecule. The assay is described in Example 3, infra.

FIG. 14 illustrates the effect of distance of the mismatches from the3′-end of a hybridized AP site probe on tail cleavage efficiency. Themismatches were placed 1, 2, 3, 4, 5, 6 and 8 bases from the 3′ end of a14-mer probe. Types of single nucleotide mismatches (SNPs) and theirpositions from the 3′-end of the probe are shown on left of each graph.The assay is described in Example 4, infra.

FIG. 15 illustrates the exquisite ability of a relatively short, 10-merAP site probe to discriminate a single nucleotide polymorphism in atargeted nucleic acid. In contrast to the longer, 14-mer probe,performance of which is shown on FIG. 14, the 10-mer probe discriminatesall studied SNPs effectively in a broad temperature range. Position of amismatch vs. the 3′-end of the probe still has an effect on the probecleavage efficiency but it is much less pronounced than for the longer,14-mer probe. The assay is described in Example 4, infra.

FIG. 16 illustrates post-PCR detection of a single nucleotidepolymorphism in human DNA samples with two 7-mer AP site probes labeledby distinguishable fluorescent dyes and containing modified “a” and “t”bases (“a” is Super A™ and “t” is Super T™, see www.Epochbio.com). Theassay is described in Example 5, infra.

FIG. 17 illustrates that cleavage of a fluorescent functional tail Rfrom an AP site probe does not effect on the probe hybridizationproperties. Melting curves of the intact and cleaved probes are shown bywhite and black dots respectively. The assay is described in Example 6,infra.

DETAILED DESCRIPTION OF THE INVENTION

I. General

The present invention provides assay methods that combine the advantagesof nucleic acid cycling despite the retained binding stability of theprobe after tail cleavage, an exquisitely target-specific enzymaticcleavage reaction, the possibility for essentially instantaneous andhighly sensitive reporter detection and the ability to directly combinedetection with amplification procedures without requiring additionalprimers, additional enzymes other than a polymerase or other additionalsteps.

II. Definitions

As used herein, an AP site probe is a nucleic acid probe comprised of anoligonucleotide sequence NA attached at its 3′ end to a phosphodiesterbond of a phosphate group, to a functional, chemical tail R comprisingan AP endonuclease cleavage site and a functional group. In preferredembodiments, the phosphate group is linked to the functional, chemicaltail through a hydroxyprolinol linker. The functional group can be areporter or a quencher group.

An abasic site is an naturally occurring Apurinic/Apyrimidinic (AP) sitein a nucleic acid sequence or a synthetic linker that is recognized andcleaved by Class II AP endonucleases when it appears in double strandedDNAs.

As used herein, an AP endonuclease refers to an enzyme that binds to andcleaves the phosphodiester backbone at an abasic (AP) site on a nucleicacid strand in a double stranded DNA. Preferred AP endonucleases cleavethe phosphodiester backbone on the 5′ side of the AP site via ahydrolytic mechanism that provides a free 3′-OH group that serves as asubstrate for DNA polymerases.

By “duplex” is intended two hybridized nucleic acid strands. A probeduplexed to a target nucleic acid, can alternately be said to behybridized to the target nucleic acid.

III. DESCRIPTION OF THE EMBODIMENTS

The present invention provides an AP site probe comprised of anoligonucleotide NA that hybridizes to a target nucleic acid, and afunctional tail R comprising a detectable reporter group and an APendonuclease cleavage site linked via a phosphodiester bond of aphosphate group to the 3′ terminal nucleotide nucleotide of the NA,wherein the reporter group is not detected when the functional, chemicaltail R is attached to the NA. The AP endonuclease preferentially cleavesthe functional tail R when the NA component is hybridized to acomplementary target nucleic acid, such that its cleavage by an APendonuclease results in a free 3′-OH group. In a preferred embodiment,the functional tail R is linked to the NA terminal 3′ phosphate groupvia a hydroxyprolinol linker. In a preferred embodiment, the reportergroup is a fluorophore. In some embodiments, an AP site probe willfurther have a quencher or quenching molecule attached to its 5′-end viaan AP endonuclease non-cleavable linker.

In some embodiments, the NA portion of the AP site probe comprises oneor more modified bases. In some embodiments, the NA portion of the APsite is a member of a universal library, usually of about 5, 6, 7 or 8nucleotides in length. In some embodiments, the NA portion of the APsite probe is a member of a universal library comprising at least onemodified base.

In an alternative embodiment, the functional tail R attached to the3′-end of the AP site probe comprises a quencher molecule attachedthrough an AP endonuclease-cleavable linker and a detectable reportergroup moiety that is attached via an AP endonuclease non-cleavablelinker to the 5′-end of the probe.

One or more AP site probes find use in methods and assays for detectinga target nucleic acid of interest in a sample. The methods involve (i)contacting the sample with at least one AP site probe and an APendonuclease, preferably a Class II AP endonuclease, under reactionconditions sufficient to allow the at least one AP site probe tohybridize to the target nucleic acid and form a reaction mixture, (ii)incubating the reaction mixture under reaction conditions that allow theAP endonuclease to cleave the phosphodiester bond attaching thefunctional tail R to the 3′ terminal nucleotide of the NA, and (iii)detecting the reporter group on the cleaved functional tail R. Themethods allow for cycling of the target nucleic acid while stillpreserving a stable hybridization complex between the NA component ofthe AP site probe and the target nucleic acid before and after cleavageof the functional tail R by the AP endonuclease.

The methods are exquisitely sensitive to the detection of single basepair mismatches between a probe NA component and a target nucleic acidbecause the AP endonuclease preferentially cleaves the phosphodiesterbond attaching the tail R to the NA when the NA is hybridized with acomplementary nucleic acid sequence in comparison to cleaving afunctional tail attached to a NA that is unhybridized or hybridized to anon-complementary nucleic acid. Usually, when carrying out a method ofdiscriminating mismatches between one or more base pairs, the targetnucleic acid sample is contacted with a first AP site probe and a secondAP site probe, where the NA component of the first probe has at leastone base difference from the NA component of the second probe, and wherethe first probe has a reporter group that is distinguishably detectablefrom the reporter group of the second probe. Preferably, the reportergroups of the first and second probe comprise a fluorophore, where thefluorophore of the first probe has a distinguishably detectable emissionwavelength from the fluorophore of the second probe. Mismatchdiscrimination is particularly sensitive when the mismatch is located atpositions 1 or 2 bases from the 3′-end of the NA component of an AP siteprobe. More than two AP site probes can be applied in the same reactionmixture to detect the target polymorphism. Those skilled in the artwould appreciate that these AP site probes would carry distinguishabledetection markers.

Usually, the target nucleic acid in a sample is further contacted withan enhancer oligonucleotide, where the 5′-end of the enhanceroligonucleotide hybridizes to the target nucleic acid on the 3′ side ofthe hybridized AP site probe, leaving a gap of 0-2 unpaired basesbetween the enhancer-target and probe-target duplexes. The mostpreferred gap is one base. In some embodiments, the AP site probe andthe enhancer oligonucleotide are attached to each other through a linkermolecule.

In some embodiments, either the target nucleic acid, the enhancer or theAP site probe are attached to a solid support. The attachment may beeither through a covalent linkage or through non-covalent interactions.

The methods for detecting a target nucleic acid of interest areparticularly suited for combining with methods of polymerase extensionof primers hybridized to the target nucleic acid. Procedures for primerextension can be carried out before or during procedures for detection.In a preferred embodiment primer extension and detection can be executeddirectly after endonuclease cleavage. Because cleavage of thephosphodiester bond of the functional tail R results in a hybridized NAhaving a free 3-OH substrate, primer extension involves further adding apolymerase and NTPs to the sample and incubating the sample underreaction conditions that allow the polymerase to extend the hybridizedNA in a template-specific manner. The methods for detecting a targetnucleic acid of interest by target-specific cleavage of the AP siteprobes are particularly suited for combining with methods of targetamplification. Target detection can be carried out during (real-time) orafter procedures for amplification. In one embodiment the AP site probecleavage detection can be executed directly after the targetamplification. In another embodiment the detection can be executedduring the target amplification. In alternative embodiments, targetamplification is isothermal amplification or polymerase chain reactionamplification.

The invention further provides primers with internal APendonuclease-cleavable sites (pL), the primers having a sequencestructure (NA₁-L)_(m)-NA₂, wherein NA₁ and NA₂ are nucleic acidsequences complementary to the target nucleic acid, L is an APendonuclease-cleavable linker, and m is from 0 to 100. The primers finduse in methods for amplifying a target nucleic acid sequence of interestin a sample, the methods involving contacting a sample with at least oneforward and at least one reverse primer having internal APendonuclease-cleavable sites, an AP endonuclease, a nucleic acidpolymerase and NTPs, under conditions sufficient to allow the forwardand reverse primers to hybridize to the target nucleic acid and form areaction mixture, and incubating the reaction mixture under reactionconditions that simultaneously allow the AP endonuclease to cleave at alinker site L, thereby generating a free 3′-OH, and the polymerase toextend the primers in a template-specific manner. The target nucleicacid can be amplified by either isothermal amplification or polymerasechain reaction.

The invention further provides kits containing reagents, including atleast one AP site probe, for carrying out the claimed methods. In kitscontaining reagents for detecting at least one single nucleotidepolymorphism, sets of 1 to 4 AP site probes are included for eachpolymorphism location. Each AP site probe in a set will have a reportergroup that is distinguishably detectable from the other AP site probereporter groups in the set intended to discriminate one or morepolymorphisms at a particular location on a target nucleic acid.

A. Target Nucleic Acid

Probes comprising a nucleic acid, an AP site and a functional tail areuseful for the detection of single-stranded nucleic acids (“ssNA”) anddouble-stranded nucleic acids (“dsNA”). When used for the detection ofdouble-stranded nucleic acids, unless the population of dsNA contains asufficient amount of ssNA to be detected using an AP site probe, thedsNA is prepared to provide a sufficient amount of ssNA. Ordinarily, thedsNA is melted or denatured at an elevated temperature prior to theirdetection. Also, dsNA can be prepared such that a fragment of the targetnucleic acids to which the probe and enhancer are complimentary issingle-stranded while the rest of the target is double-stranded.Single-stranded target nucleic acids can be isolated from thedouble-stranded forms using available molecular biology orphysicochemical methods, including strand-specific enzymaticdegradation, limited digestion of the double-stranded target followed byheat treatment, or affinity capture through a sequence-incorporatedaffinity label followed by heat-induced separation from thecomplementary strand.

Target nucleic acids can be isolated from a variety of natural sources,including blood, homogenized tissue, fixed tissue, tumor biopsies,stool, clinical swabs, food products, hair, plant tissues, microbialculture, public water supply, amniotic fluid, urine, or the like.Techniques useful for the isolation of target nucleic acids include, forexample, amplification techniques, e.g., polymerase chain reaction(PCR), Mullis, U.S. Pat. No. 4,683,202; ligase-based techniques, e.g.,reviewed by Barany, PCR Methods and Applications 1: 5-16 (1991);strand-displacement amplification, Walker et al., U.S. Pat. No.5,422,252; reverse transcriptase-based techniques, e.g., Davey et al.,U.S. Pat. No. 5,409,818; Q.beta. replicase-based techniques, e.g., Chuet al., U.S. Pat. No. 4,957,858; branched DNA techniques, Urdea et al.,U.S. Pat. No. 5,124,246; techniques employing RNA-DNA chimeric probes,Duck et al., U.S. Pat. No. 5,011,769; and the like.

Samples containing target nucleic acids can be isolated from naturalsources or provided as result of any known method in the art. The targetnucleic acid can be cloned, synthetic, or natural. The target nucleicacid can be deoxyribonucleic acid (DNA), including genomic DNA or cDNA,or ribonucleic acid (RNA). Usually a DNA target nucleic acid ispreferred. Target nucleic acids can be of diverse origin, includingmammalian, bacterial, fungal, viral, or plant origin. The need forextraction, purification, or isolation steps depends on several factors,including the abundance of the target nucleic acids in the sample, thenature of the target nucleic acids, e.g., whether it is RNA or DNA, thepresence of extraneous or associated material such as cell walls,histones, or the like, the presence of enzyme inhibitors, and so forth.

Guidance for selecting an appropriate protocol for particularapplications for extraction, purification and/or isolation of targetnucleic acids can be found in, for example, Chen and Janes, Editors, PCRCloning Protocols (Humana Press, Totowa, N.J., 2002); Sambrook et al.,Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory Press,2001); White, Editor, PCR Cloning Protocols: from molecular cloning togenetic engineering (Humana Press, Totowa, N.J., 1997); Methods inEnzymology, Volumes 6 and 12, parts A and B (Academic Press, New York);McPherson et al., Editors, PCR: A Practical Approach (IRL Press, Oxford,1991); Herrington et al., Editors, Diagnostic Molecular Pathology: APractical Approach, Vol. 1 & 2 (IRL Press, Oxford, 1992); Innis, et al.,Editors, PCR Protocols (Academic Press, San Diego, 1990); and the like.Typically, preparation protocols involve the application of chaotropicagents, for example, low molecular weight ionic compounds, that favorthe solubilization of hydrophobic substances, chelating agents (forinstance, EDTA), to disable nucleases, proteases to disable nucleases,detergents, pH buffers, and the like, that serve to isolate and/orprotect nucleic acids. Optionally, samples can be treated to reduce thesize of the target nucleic acids, such as by sonication, nucleasetreatment, or the like. After such initial preparation steps, preferablya sample is treated to denature, i.e. render single-stranded, the targetpolynucleotide prior to exposing it to the nucleic acid probe, enhancerand AP endonuclease in accordance with the invention. Preferably,denaturation is achieved by heating the sample at 93-95° C. for fiveminutes.

In assays of the present invention, a target nucleic acid is typicallyincluded in concentrations of about 2-10 nM, more typically about 4-8nM, and preferably at a concentration of about 5 nM. However, one ofskill in the art will appreciate that the invention is not so limitedand other concentrations of target can also be used, whether higher orlower than those indicated above.

B. AP Site Probe

Generally, the structure of an AP site probe is as follows:

An AP site probe is comprised of a nucleic acid (“NA”) covalently boundby its 3′-terminal oxygen atom to a functional, chemical tail (“R”)through a phosphodiester group.

1. Nucleic Acid Component of Probe

The number of nucleotides in the NA component can be 3 to 200, 3 to 100or 3 to 200 nucleotides in length, depending on the intended use.Usually, the length of the NA is from 5 to 30 nucleotides. Moretypically, the length of the NA is 6-25, 7-20, or 8-17 nucleic acids.Most often, the NA component is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or-16 nucleic acids in length. Usually, the NA component will have ahybridization melting temperature of about 10 to 80° C., more typicallyof about 20 to 70° C., and preferably about 30° C., 40° C., 50° C. or60° C.

The sugar, or glycoside, portion of the NA component of the conjugatescan comprise deoxyribose, ribose, 2-fluororibose, and/or 2-O-alkyl oralkenylribose wherein the alkyl group comprises 1 to 6 carbon atoms andthe alkenyl group comprises 2 to 6 carbon atoms. In thenaturally-occurring nucleotides, modified nucleotides and nucleotideanalogues that can comprise an oligonucleotide, the sugar moiety forms afuranose ring, the glycosidic linkage is of the beta configuration, thepurine bases are attached to the sugar moiety via the purine 9-position,the pyrimidines via the pyrimidine 1-position and thepyrazolopyrimidines via the pyrazolopyrimidine 1-position (which isequivalent to the purine 9-position). In a preferred embodiment, thesugar moiety is 2-deoxyribose; however, any sugar moiety known to thoseof skill in the art that is compatible with the ability of theoligonucleotide portion of the compositions of the invention tohybridize to a target sequence can be used.

In one preferred embodiment, the NA is DNA. An AP site probe comprisingDNA can be used to detect DNA, as well as RNA, targets. In anotherembodiment, the NA is RNA. An AP site probe comprising RNA is generallyused for the detection of target DNAs. In another embodiment, an AP siteprobe can contain both DNA and RNA distributed within the probe. Inmixed nucleic acid probes, DNA bases preferably are located at 3′-end ofthe probe while RNA bases are at the 5′-end. It is also preferred whenthe 3′-terminal nucleotide is 2′-deoxyribonucleotide (DNA) and when atleast four 3′-terminal bases of NA are DNA bases.

Usually, the NA component contains the major heterocyclic basesnaturally found in nucleic acids (uracil, cytosine, thymine, adenine andguanine). In some embodiments, the NA contains nucleotides withmodified, synthetic or unnatural bases, incorporated individually ormultiply, alone or in combination. Preferably, modified bases increasethermal stability of the probe-target duplex in comparison with probescomprised of only natural bases (i.e., increase the hybridizationmelting temperature of the probe duplexed with a target sequence).Modified bases include naturally-occurring and synthetic modificationsand analogues of the major bases such as, for example, hypoxanthine,2-aminoadenine, 2-thiouracil, 2-thiothymine, inosine,5-N⁴-ethenocytosine, 4-aminopyrrazolo[3,4-d]pyrimidine and6-amino-4-hydroxy-[3,4-d]pyrimidine. Any modified nucleotide ornucleotide analogue compatible with hybridization of an AP site probewith a target nucleic acid conjugate to a target sequence is useful inthe practice of the invention, even if the modified nucleotide ornucleotide analogue itself does not participate in base-pairing, or hasaltered base-pairing properties compared to naturally-occurringnucleotides. Examples of modified bases are disclosed in U.S. Pat. Nos.5,824,796; 6,127,121; 5,912,340; and PCT Publications WO 01/38584; WO01/64958, each of which is hereby incorporated herein by reference inits entirety. Preferred modified bases include 5-hydroxybutynyl uridinefor uridine;4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol,4-amino-1H-pyrazolo[3,4-d]pyrimidine, and4-amino-1H-pyrazolo[3,4-d]pyrimidine for adenine;5-(4-Hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione for thymine; and6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one for guanine. Particularlypreferred modified bases are “Super A™,” “Super G™: 4-hydroxy-6-aminopyrazolopyrimidine” (www.Epochbio.com) and “Super T™”. Modified basespreferably support the geometry of a naturally occurring B-DNA duplex.Modified bases can be incorporated into any position or positions in anAP site probe, but preferably are not incorporated as the 3′-terminalbase.

In another embodiment, some or all nucleotides of NA are substituted orcontain independently different sugar-phosphate backbone modificationsincluding 2′-O-alkyl RNA nucleotides, phosphorotioate internucleotidelinkage, methylphosphonate, sulfamate (e.g., U.S. Pat. No. 5,470,967)and polyamide (i.e., peptide nucleic acids, PNA), LNA (locked nucleicacid), and the like. Such modifications and others of potential use inthe present invention are described, for example, in Boutorine, et al.,Biochimie 76:23 (1994); Agrawal, et al., Proc. Natl. Acad. Sci. 88:7595(1991); Mag, et al., Nucleic Acids Res. 19:1437 (1991); Kurreck, Eur. JBiochem. 270:1628 (2003); Lesnik, et al., Biochemistry 32:7832 (1993);Sproat, et al., Nucleic Acids Symp. Ser. 24:59 (1991); Iribarren, etal., Proc. Natl. Acad. Sci. 87:7747 (1990); Demidov, Trends Biotechnol.21:4 (2003); Nielsen, Methods Mol. Biol. 208:3 (2002); Nielsen andEgholm, Curr. Issues Mol. Biol. 1:89 (1999); Micklefield, Curr. Med.Chem. 8:1157 (2001); Braasch, et al., Chem. Biol. 8:1 (2001); andNielsen, Curr. Opin. Biotechnol. 12:16 (2001).

Within the scope of present invention, modifications of the bases andsugar-phosphate backbone as well as other functional moieties conjugatedwith the probe can serve to improve the sequence specificity of thetarget-probe duplex formation. In particular, binding between the probeand a matched target nucleic acid is detectably increased over bindingto a mismatched target nucleic acid. By “matched target nucleic acid” isintended a target nucleic acid that contains a sequence that iscompletely complimentary to the probe sequence. By “mismatched targetnucleic acid” is intended a polynucleotide that contains a sequence thatis partially complimentary to the probe sequence such that it containsat least one mismatched, non-complimentary base, deletion or insertionin comparison to the probe sequence. For example, use of modified basesin an AP site probe allows for more stable base pairs than when usingnatural bases and enables the use of shorter probes for the samereaction conditions. Reduction of the probe length increases the abilityof the probe to discriminate a target polymorphism as small as a SingleNucleotide Polymorphism (“SNP”) due to a proportional increase in thecontribution of each duplex base pair to the overall duplex stability.In general, the shorter the probe, the greater the relative contributionof an individual base pair in to the overall duplex stability, and thebetter the probe discrimination of the target polynucleotidepolymorphism.

2. Functional Tail (“R”) Component of Probe

The functional tail R enables detection of the endonucleasetail-cleavage reaction. The structure of R can be of any size andcomposition as long as it supports the template-specific, endonucleasetail-cleavage reaction. R can be as large as a natural protein withmolecular mass up to 1,000,000 Daltons or it can be as small as a singleatom (i.e., a radioactive isotope, such as a hydrogen or an iodine).Since the enzymatic hydrolysis occurs between the 3′-terminal oxygenatom of the NA and the phosphorus atom of the phosphodiester bond, forthe purposes of the present invention, the phosphate moiety of the probeis considered a part of the functional tail R. For example, when R ishydrogen (R═H), the functional tail of the probe is a phosphate moiety—P(O)(OH)₂ or —PO₃ ²⁻. The tail R can be hydrophobic or hydrophilic,electrically neutral, positively or negatively charged. It can becomprised of or include independently different functional groups,including mass tags, fluorescent or non-fluorescent dyes, linkers,radioisotopes, functional ligands like biotin, oligopeptides,carbohydrates and the like. For example, as demonstrated herein,Endonuclease IV from E. coli efficiently cleaves from the 3′-end of aprobe bound to the target nucleic acid a relatively hydrophilic,negatively charged fluorescein moiety as well as an electricallyneutral, hydrophobic quenching dye.

The tail R can contain components that improve specificity by blockingnon-specific cleavage reactions in the absence of a target moleculewithout affecting the target-dependent, specific reaction. It is alsowithin the scope of present invention that the tail R or some structuralcomponents of it can improve the specificity of the target-probe orenhancer-probe complementary binding so that the thermodynamicdifference in the probe/enhancer binding to matched and mismatchedtarget nucleic acids is increased. Examples of such structuralcomponents are minor groove binders (MGBs).

The functional tail R can incorporate mono-, oligo- or polynucleotides.Nucleotide residues introduced into the tail structure are not intendedto bind to the target nucleic acid.

In addition to a functional chemical tail R conjugated to the 3′-end ofan AP site probe through a phosphodiester group, the probe optionallycan contain other tails and functional moieties covalently attached tothe probe or the tail via an appropriate linker. Preferably, theadditional moieties do not interfere with endonuclease recognition ofthe AP tail-cleavage site or the template-specific tail-cleavagereaction. In one embodiment, additional moieties are attached to the5′-end of the NA portion of the probe. In another embodiment, anadditional moiety is conjugated to nucleotide bases of the probe suchthat, when the probe-target duplex is formed, the moieties are locatedwithin the major groove of the duplex.

Incorporation of a moiety in addition to the functional, chemical tailcan serve to improve the probe hybridization properties. Examples ofsuch moieties include minor groove binders and intercalators. Minorgroove binders are described in U.S. Pat. Nos. 6,492,346 and 6,486,308,both of which are hereby incorporated herein by reference. In otherembodiments, these moieties operate in conjunction with the functionaltail R to aid in the detection of an endonuclease tail-cleavagereaction. Examples of such moieties include radioisotopes, radiolabelledmolecules, fluorescent molecules or dyes, quenchers (dyes that quenchfluorescence of other fluorescent dyes), fluorescent antibodies,enzymes, or chemiluminescent catalysts. Another suitable moiety is aligand capable of binding to specific proteins which have been taggedwith an enzyme, fluorescent molecule or other detectable molecule (forexample, biotin, which binds to avidin or streptavidin, or a heminmolecule, which binds to the apoenzyme portion of catalase).

In a preferred embodiment, both the functional tail R and the additionalmoiety are dyes. One or both of the tail and additional moiety dyes canbe fluorescent dyes. Preferably, one of the dyes is fluorescent. In onepreferred embodiment the functional tail comprises a fluorescent dye andthe additional moiety comprises a quencher. The fluorescent dye andquencher molecule operate together such that the fluorescence of the dyeis repressed when the dye is bound to the AP site probe, but thefluorescence of the dye is detectable when the phosphodiester bondbetween the NA and tail R is hydrolyzed or cleaved by the enzyme. Thisfluorescence detection strategy is known as Fluorescence ResonanceEnergy Transfer (FRET). According to a FRET technique, one of the dyesservers as a reporter dye and the other dye is a quencher thatsubstantially decreases or eliminates fluorescence of the reporter dyewhen both of the dyes are bound to the same molecule in proximity ofeach other. The fluorescence of the reporter dye is detected whenreleased from the proximity of the quencher dye. Cleavage of the AP siteprobe functional tail releases the reporter dye from its quenchercounterpart allowing for a detectable increase in the reporterfluorescence and detection of the target nucleic acids. The quenchingdye can be a fluorescent dye or non-fluorescent dye (dark quencher).See, U.S. patent Publication Ser. No. 2003/0,113,765, U.S. Ser. No.2003/0,096,254 and PCT Publication No. WO 01/42505 for fluorophore andquencher examples, both of which are hereby incorporated herein byreference.

The present invention includes a composition comprising a solid supportand an AP site probe immobilized thereon. In such a case, one of themoieties conjugated to the probe can be a moiety that serves to attachthe probe to the solid support. This moiety or solid support linker canbe attached anywhere within or be a structural part of the NA andfunctional tail R structures of the probe of the present invention. Inone embodiment, the AP site probe is covalently attached to a solidsupport through a Schiff base type linkage, as described in U.S. Pat.No. 6,548,652, incorporated herein by reference.

In assays of the present invention, a probe is typically included atconcentrations of about 50-200 nM, more typically at concentrations ofabout 100-175 nM, and preferably at concentrations of about 150 nM. Oneof skill in the art will appreciate that the probe concentrationsprovided above can be altered depending on a variety of factors,including the amount of target, as well as the characteristics of thedye or quencher used.

C. Enhancer

An enhancer is an oligo- or polynucleotide designed to form a duplexwith the target nucleic acid positioned immediately 5′- to the target-APsite probe. The combined, probe-enhancer-target complex simulates anaturally occurring nucleic acid atypical abasic site that is recognizedby cellular exo- and endonuclease repair enzymes. Although the tail Rcleavage reaction can be achieved without the enhancer, the presence ofan enhancer generally improves the kinetics the reaction.

The structural requirements and limitations for an enhancer areessentially the same as for a NA component of an AP site probe,described above. Generally, the number of nucleotides in an enhanceroligonucleotide can range from 3 to 50, 100 or 200 nucleotides inlength. Usually, the length of an enhancer is from 5 to 30 nucleotides.More typically, the length of the enhancer is 6-25, 7-20, or 8-15nucleic acids. Most often, an enhancer component is about 10, 12, 14,16, 18 or 20 nucleic acids in length. Usually, an enhanceroligonucleotide component will have a hybridization melting temperatureof about 10 to 80° C., more typically of about 20 to 70° C., andpreferably about 30° C., 40° C., 50° C., 60° C. or 70° C. An enhanceroligonucleotide will usually have a comparatively equal or higherhybridization melting temperature in comparison to the meltingtemperature of the NA component of the AP site probe. Usually, themelting temperature will be about 5 to 30° C., more typically about 10to 20° C., and preferably about 8° C., 10° C., 15° C., or 20° C. higherthan the melting temperature of the NA component of the AP site probe.

Preferably, the enhancer is DNA. An oligo- or polydeoxyribonucleotideenhancer is useful for detecting DNA and RNA target nucleic acids. Theenhancer can also be RNA. In another embodiment, an enhancer can containboth DNA and RNA. Preferably, DNA bases are located at the 5′-end of theenhancer while RNA bases are at its 3′-end. Preferably, at least thefour 5′-terminal bases of the enhancer are DNA bases.

In another embodiment, the enhancer contains nucleotides with modified,synthetic or unnatural bases, including any modification to the base,sugar or backbone. Preferably, modified bases increase thermal stabilityof the enhancer-target duplex in comparison to enhancer sequences thatcontain only natural bases. Specific modified bases are the same asthose described for a probe.

In another embodiment, some or all nucleotides of the enhancer aresubstituted or contain independently different sugar-phosphate backbonemodifications, including, 2′-O-alkyl RNA nucleotide, phosphorotioateinternucleotide linkage, PNA (peptide nucleic acid), LNA (locked nucleicacid). References describing these and other potentially usefulsugar-phosphate backbone modifications are provided above.

The enhancer optionally can contain some functional tails or markersconjugated to either end of the enhancer or in the middle of it. Thesemoieties should not interfere with the template-specific cleavage of theprobe R tail. In a preferred embodiment, these moieties are attached tothe 3′-end of the enhancer. In another preferred embodiment, thesemoieties are conjugated to nucleotide bases of the enhancer such that,when the enhancer-target duplex is formed, the moieties are locatedwithin the major groove of this duplex. Enhancer moieties can serve toimprove the enhancer hybridization properties. Examples of such moietiesinclude minor groove binders and intercalators.

The present invention also encompasses a composition comprising anenhancer immobilized on a solid support. A moiety conjugated to theenhancer can serve to attach the enhancer to the solid support. Thismoiety or solid support linker can be attached anywhere within or be astructural part of the enhancer.

Modifications of the bases and sugar-phosphate backbone as well as otherfunctional moieties conjugated to the enhancer can serve to improve thesequence specificity of target-enhancer duplex formation resulting inincreased thermodynamic differences in binding between the enhancer anda matched target nucleic acid in comparison to binding between theenhancer and a mismatched target nucleic acid.

In assays of the present invention, an enhancer, when included, istypically added at concentrations of about 50-200 nM, more typically atconcentrations of about 100-175 nM, and preferably at concentrations ofabout 150 nM.

D. Enzyme

An enzyme used in the present invention is an endonuclease orexonuclease that recognizes an Apurinic/Apyrimidinic (AP) site oratypical AP site moiety simulated by an AP site probe duplexed with atarget nucleic acid complex, and preferentially hydrolyzes or cleavesthe phosphodiester bond between the probe and the functional tail R. Anenhancer can be used to increase the kinetics of the tail-cleavagereaction. An enzyme useful in the present methods preferentially doesnot cleave the NA part of the probe or the target nucleic acid.Otherwise, enzymes which cleave the probe NA or target nucleic acid atan efficiency that is substantially lower than target-specific tailcleavage can still find use in practicing the present methods. Tominimize non-specific detection of the target nucleic acid, the enzymepreferentially does not cleave the tail R of the probe in absence of thetarget nucleic acid.

In a preferred embodiment, the enzyme is an AP endonuclease. The enzymecan be a class I or a class II AP endonuclease. Preferably, the enzymeis a class II endonuclease. Enzymes that belong to this family areisolated from variety of organisms, and any class II endonuclease thatspecifically recognizes an AP abasic site and specifically hydrolyzesthe phosphodiester backbone on the 5′ side of the AP site can be used inthe present methods. Exemplified class II AP endonucleases includeEndonuclease IV and Exonuclease III from E. coli, human APEI/REF-1endonuclease, yeast APN1 endonuclease, exonuclease III homologousenzymes from Drosophila (Rrp1) and Arabidopsis (Arp) and thermostableendonuclease IV from Thermotoga maritima. Other AP endonucleases usefulfor detection and/or amplication systems requiring an AP site probe canbe identified through the National Center for BiotechnologicalInformation Entrez/PubMed nucleotide and protein databases accessedthrough the website www.ncbi.nlm.nih.gov/. Enzymes homogolous instructure and function to the E. coli Exonuclease III family of APnucleases are also of use in the present invention (Mol, et al., Mutat.Res. 460:211 (2000); Ramotar, Biochem. Cell Bio. 75:327 (1997)). Thestructure and function of apurinic/apyrimidinic endonucleases isreviewed by Barzilay and Hickson in Bioessays 17:713 (1995).

In a preferred embodiment, the enzyme is an E. coli Endonuclease IV. AnE. coli Endonuclease IV exhibits catalytic activity between roomtemperature (25° C.) and 75° C., preferably between 40-70° C. or 40-60°C., and more preferably between 60-70° C. or 65-75° C. The temperatureof a target nucleic acid detection assay is preferably determined by thehybridization melting temperature of an AP site probe, where thetemperature of the reaction conditions is preferably within 5, 4, 3, 2,1 or 0 degrees, above or below, of the probe melting temperature, T_(m).Optimum catalytic activity of an Endonuclease IV is observed within a pHrange of 7.5-9.5, preferably between pH 8.0-9.0, most preferably atabout pH 8.5-9.0. An abasic site assay using an Endonuclease IV enzymeis preferably carried out using a buffer that maintains a steady pHvalue of between 7.5-9.5 over varying temperatures. Preferred buffersinclude HEPPS (4-(2-hydroxyethyl)-1-piperazinpropan-sulfonic acid) andHEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid). In apreferred embodiment, the buffer used is HEPPS-KOH. In certainembodiments, a TRIS buffer is also appropriate. Additional biologicalbuffers of potential use can be found through Sigma-Aldrich (St. Louis,Mo., www.sigma.com). Usually, the reaction conditions contain enzyme innanomolar concentrations, but tail cleaving activity can be observedwhen the enzyme is provided in picomolar concentrations, and in certaincases in femtomolar concentrations.

E. Positioning of the Probe and Enhancer Binding Sites

FIG. 2 shows an optimal design of the probe and enhancer to achieve thehighest yield of the tail-cleavage reaction. The probe and enhancer formduplexes with the target nucleic acid that are positioned next to eachother leaving one, non-paired base of the target between the duplexes.This design simulates the naturally occurring lesion 2 that is shown inFIG. 1. Although this is a preferred design, cleavage of the tail R inthe target-probe complex can be achieved in absence of the enhancer, orwhen the number of non-paired, target polynucleotide bases between twoduplexes shown is 0, 2 or more bases. All these designs are within thescope of the present invention.

Including an enhancer can be desirable, especially when using an enzymeof the E. coli Endonuclease IV family, because the enzyme tail-cleavagerate, as measured by detectable reporter signal, can be increased 6, 7,8, 9, or 10 fold in comparison to the tail-cleavage rates in the absenceof an enhancer.

F. Cycling of the Tail Cleavage Reaction

In the past, probes used in cycling probe assays have typicallypositioned a cleavable linker somewhere within the middle of a probesequence. This design is believed to provide a strong thermodynamicfactor to drive the cycling process when the target polynucleotide isrecycled during the reaction. Cleavage of the probe within the middle ofthe nucleotide sequence leads to products that are shorter in length andthat have weaker hybridization properties than the intact probe. Atoptimal reaction conditions that are typically below the probe T_(m),the product-target complexes fall apart, quickly recycling the targetnucleic acid for binding with other intact probe molecules.

The probe design in the present invention lacks a thermodynamic,cycling-driven factor. The hybridization properties of the probe remainessentially the same before and after the tail cleavage reaction. SeeFIG. 17. An AP site probe having a cleavable functional tail at the 3′end of the probe also supports a cycling mechanism. The target nucleicacid remains intact after tail cleavage and is available to bind anotherAP site probe having a cleavable functional tail. Typically, the numberof the cleaved probes per target molecules is greater than one, moretypically about 5, 10, 20, or 30, and can be as many as 40 or 50.Without being bound to any particular theory, the cycling observedherein appears to be “kinetically driven” in contrast to“thermodynamically driven” cycling disclosed by others and it isconceivably the result of several factors. First, when the reactiontemperature is close to the probe hybridization melting temperature suchthat the lifetime of the probe-target complex is relatively short, itleads to a rapid exchange of the probe molecules in the probe-targetduplex. As a general rule, the closer the reaction temperature is to theprobe T_(m), the faster the cycling. Second, when the tail-ON probeconcentration is in excess over the tail-OFF product, for instance, atthe earlier stages of the reaction, the tail-ON probe is predominantlysupplied to the reaction complex, facilitating cycling. It isunderstood, within the scope of present invention, that an optimalreaction temperature of the AP site probe cleaving assay, a temperatureat which the observed cleavage rate is maximum, can be different fromthe melting temperature of the AP-site probe. It can be lower or higherthan the AP probe T_(m). This is due to factors effecting the AP siteprobe cleavage reaction. Examples of these factors are AP endonucleaseactivity at different temperatures, elements of secondary structureswithin the nucleic acid components of the reaction, target nucleic acid,AP site probe and enhancer that compete with the formation of thedesired active complex (see FIG. 2). Finally, the Endonuclease canpreferentially bind to and stabilize the tail-ON probe-target nucleicacid duplex over the tail-OFF complex, promoting the cycling process.

G. Detection of the Endonuclease Tail-Cleavage Reaction

Either part of the endonuclease tail-cleavage reaction, the NAcontaining part or the tail R containing part or alternatively both ofthem independently, can be detected. Suitable reporter groups forattaching to the functional tail R include beads, nanoparticles (Taton,et al., Science 289:1757 (2000), chemiluminescers, isotopes, enzymes andfluorophores. A variety of physical or chemical methods can be used fordetection of the cleavage product. Depending on the nature of themarkers used, these methods include, for example, chromatography andelectron-, UV-, IR-, mass-, radio-, fluorescence spectroscopy includingfluorescence polarization and the like.

In a preferred embodiment, cleavage of the functional tail R comprises afluorophore reporter group and is detected by fluorescence spectroscopy.Suitable fluorophores include the resorufin dyes, coumarin dyes,xanthene dyes, cyanine dyes, BODIPY dyes and pyrenes. Preferably, thefunctional tail R comprises a fluorescent dye with a xanthene corestructure. Exemplified dyes with a xanthene core structure are depictedin FIG. 7. Additional fluorophores appropriate for incorporation intothe functional tail R are described in PCT Publication No. WO 01/142505and in Haugland, Handbook of Fluorescent Probes and Research Products,Ninth Ed., (2002), published by Molecular Probes, Eugene, Oreg.(accessible at www.probes.com/handbook/).

In some embodiments, background fluorescence of a fluorophoreincorporated on the functional tail R, is minimized by attaching aquencher to the AP site probe. Typically, a quenching molecule iscovalently attached to the 5′ end of the probe through a linker that isnot cleaved by an enzyme. In some embodiments, a quencher is linked tothe middle or the 3′ end of the probe. When a quencher is attached tothe 3′ end of the probe, it is usually incorporated into the functionaltail R as a “cleavable quencher,” and the fluorophore is then attachedto the middle or the 5′ end of the probe. In preferred embodiments thequencher comprises a dye core structure shown in FIG. 8. However, anymolecule that neutralizes or masks the fluorescence of a fluorophoreincorporated in an uncleaved functional tail R finds use as a quencherin the present invention. Other quencher molecules suitable to attach toan AP site probe and guidance for selecting appropriate quencher andfluorophore pairs is provided in Haugland, supra. Additional guidance isprovided in U.S. Pat. Nos. 3,996,345 and 4,351,760, and U.S. PublicationSer. Nos. 2003/0096,254 and 2003/0113,765 and in co-owned U.S. patentapplication Ser. No. 09/457,616, filed on Dec. 8, 1999, each of which ishereby incorporated herein by reference.

Fluorophore and cleavable quencher molecules are typically attached toan AP site probe through a linker that is specifically cleaved by anenzyme. A linker can be rigid or flexible. Preferably the linkerstructurally mimics a naturally occurring abasic site (see, FIG. 9), andis cleaved by an Endonuclease IV. Preferably the C1 carbon of thelinker, attached to the phosphate, is a primary carbon. Preferably thelinker comprises a phosphate. Exemplified linkers for attaching afluorophore or cleavable quencher molecule to a AP site probe aredepicted in FIGS. 7 and 8. Suitable commercially available chemicallinkers can be purchased through Pierce Biotechnology, Rockford, Ill.and Molecular Probes, Eugene, Oreg. Suitable methods for attachingreporter groups such as fluorophores and quenchers through linkers tooligonucleotides are described in, for example, U.S. Pat. Nos.5,512,677; 5,419,966; 5,696,251; 5,585,481; 5,942,610 and 5,736,626,each of which are hereby incorporated herein by reference.

In a preferred embodiment the linker is a rigid linker. In one preferredembodiment, the rigid linker is a hydroxyprolinol linker, such as isdepicted in FIG. 9. Hydroxyprolinol linkages are described in U.S. Pat.Nos. 5,419,966; 5,512,677; 5,519,134; and 5,574,142 each of which isincorporated herein by reference. Cleavage of the functional tail Rattached through a rigid linker, i.e., a hydroxyprolinol linker,requires greater concentrations of enzyme and exhibits decreasedcatalytic rates, but is highly specific. Generally, the Endonuclease IVenzyme does not detectably cleave functional tails R attached to an APsite probe through a rigid linker, such as a hydroxyprolinol linker, inthe absence of a target nucleic acid.

In some embodiments, it is desirable to attach the functional tail Rthrough a flexible linker. Cleavage of the functional tail R is moreefficient when attached through a flexible linker, however, decreasedspecificity is observed because detectable tail-cleavage occurs in theabsence of a target nucleic acid. Non-specific cleavage of functionaltails R attached through a flexible linker can be minimized by adding acompetitive binding substrate that is more favorable to the enzyme thanan unduplexed probe but less favorable than the probe duplexed with atarget nucleic acid, i.e., a “decoy.” In one embodiment unmelted genomicDNA is added to the reaction as a decoy to minimize cleavage of the APsite probe functional tail R in the absence of a target nucleic acid.

The ability of particular tail structures to serve as specificsubstrates of an AP endonuclease can be determined using an assay thatprovides a probe/target nucleic acid/enhancer complex as a singlehairpin structure, exemplified in FIG. 10. Preferably the hairpinstructure has one unpaired nucleic acid, thereby simulating a naturallyoccurring abasic site residing in duplexed nucleic acids. In otherembodiments, the test assay hairpin structure can have zero or twounpaired nucleic acids. In such a test assay, the cleavage of thefunctional tail R is detected by measuring the release of the reportergroup attached to a hairpin structure in comparison to release of thereporter group attached to an unduplexed AP site probe. A tail structurethat serves as a specific substrate for an AP endonuclease will becleaved from a hairpin structure at a faster catalytic rate incomparison to its cleavage rate from an unduplexed AP site probe. A tailstructure that serves as a specific substrate preferably exhibits aratio of specific cleavage, in the presence of the hairpin structure, tonon-specific cleavage, in the presence of an unduplexed AP site probe,of at least 50-, 75-, or 100-fold, more preferably of 300-, 400-, 500-,600-, 700-, 800-, 900- or 1000-fold, and can exhibit ratios of greaterthan 1000-fold, as measured by the reporter group signal (i.e.,Fluorescence Units per minute of a fluorphore reporter group). Thehairpin substrate design exemplified on FIG. 10A does not incorporate aquencher moiety. Nevertheless AP endonuclease cleavage of thefluorescent tail increases the dye fluorescence by approximately twotimes (FIG. 10B). The fluorescent signal outcome of the assay can beimproved by incorporation of a quenching moiety within the hairpinsequence that represents an enhancer. Those skilled in the art willappreciate that the hairpin substrate exemplified in FIG. 10 can be usedfor detection as well as for quantitative measurement of AP endonucleaseactivity in different media.

In other embodiments, the NA part of the AP site probe is detected. Forinstance, the products of the probe tail-cleavage reaction can bedetected as a result of another reaction that follows the cleavagereaction or occurs simultaneously with it. Cleavage of the tail R fromthe probe generates a “free” 3′-hydroxyl group that can be, for example,extended by a polymerase in a template-dependent polynucleotidesynthesis in the presence of NTPs such that the tail-OFF probe wouldserve as a primer complexed with template. In some embodiments, thestrands of a probe extension nucleotide synthesis are the detectablereaction product. Some NTPs incorporated in a probe extension canoptionally carry a detectable marker. Incorporation of one or moredetectable markers into a probe extension product simplifies thedetection of the synthesized nucleotide strands.

The excess unincorporated NTPs carrying a detectable marker need to beremoved from the reaction mixture in order to detect the synthesizedstrands of the probe extension. This can be achieved when the reactioncomplex shown on FIG. 2 is immobilized on a solid support. The complexcan be immobilized before or after the combined tail cleavage/probeextension reaction is completed. A schematic diagram of such an assay isshown on FIG. 3. Although immobilization is an effective way to removethe excess of the labeled NTPs, labeled NTPs can also be removed insolution phase. An example of such an approach is shown on FIG. 4. Ofcourse, inclusion of an enhancer is optional in AP site probe extensionor amplification assays.

H. Solid Support Tail Cleaving Assay

The target nucleic acid-probe-enhancer complex can be covalentlyattached to a solid support via a linker or linkers coupled to one, orindependently to two, components of the probe-target-enhancer complex.Immobilization of the complex also can be achieved through non-covalentbinding, including affinity, charge or hydrophobic interaction.Immobilization can be performed before or after the tail cleavingreaction. The solid support material can be, for example, latex,plastic, derivatized plastic, polystyrene, magnetic or non-magneticmetal, glass or silicon surface or surfaces of test tubes, microtiterwells, sheets, beads, microparticles, chips, and other configurationsknown to those of ordinary skill in the art. Such materials can be usedin suitable shapes, such as films, sheets and plates, or they can becoated onto or bonded or laminated to appropriate inert carriers, suchas paper, glass, plastic films, or fabrics.

I. Probe and Enhancer Bound Together Through a Linker

In one embodiment, a probe is linked to an enhancer so as these twocomponents of the reaction complex are associated with each other duringthe tail cleaving reaction. The linker can be a covalent or anon-covalent linker, i.e., when interaction between a probe and enhanceris provided by hydrogen bonds or Van der Waals forces. A probe-enhancerlinker can be attached at any position within the probe and enhancer.Preferably, the linker does not block the tail cleaving reaction, and isof an appropriate length to support the tail cleaving reaction. Further,a linker useful in a tail cleaving assay will not compromise the abilityof the AP site probe or enhancer to form duplexes with a target nucleicacid. Finally, a preferred linker is not cleaved by an AP endonuclease.FIG. 5 schematically depicts two possible arrangements of linkersbetween a probe and an enhancer. When attached through a linker, theprobe and enhancer are components of one molecule or complex. Linkedprobe-enhancer molecules or complexes can be immobilized on a solidsupport.

In preferred embodiments, a probe-enhancer linker is comprised ofindividual or combined repeats of substituted alkyl backbone moieties,including (—OCH₂CH₂—)_(n), (—OCH₂CH₂—OPO₂—)_(n) or —O(CH₂)_(n)O—.Typically, n is from 1-100, more typically n is 10, 20, 40, 50, 60 or80. In other embodiments, a linker is a flexible polypeptide chain, forinstance, dihydropyrroloindole peptides or a series of one or morerepeats of a Gly-(Ser)₄ (SEQ ID NO:2) polypeptide sequence. In anotherembodiment, the linker is an oligonucleotide, such as poly A or poly Tand the like. In yet another embodiment, the linker is an alkyl chainhaving a backbone typically of about 100, 200 or 300 atoms, moretypically of about 40, 60 or 80 atoms. Other alkyl linkers of potentialuse are described in U.S. Patent Publication No. 2003/0113765,incorporated herein by reference. Additional linkers that may find useare described by Dempey, et al., Nucleic Acids Res. 27:2931 (1999);Lukhtanov, et al., Nucleic Acids Res. 25:5077 (1997); Lukhtanov, et al.,Bioconjug. Chem. 7:564 (1996); and Lukhtanov, et al., Bioconjug. Chem.6:418 (1995). Appropriate linkers can be obtained from commerciallyavailable sources, for example from Pierce Biotechnology, Rockford Ill.(www.piercenet.com/). Guidance for selecting an appropriate linker forattaching oligonucleotides is provided in Haugland, Handbook ofFluorescent Probes and Research Products, supra. These linkers also findapplication in attaching an AP site probe or an enhancer to a solidsupport.

IV. Applications of AP Endonuclease Tail-Cleavage Systems

A. Amplification of Nucleic Acids Using Primer Cleaving Technology

An AP site probe can also function as a primer and its use in detectionof nucleic acid sequences can be combined with amplification techniquesin several ways. Amplification can be carried out before orsimultaneously with cleaving the functional tail R from an AP siteprobe.

In one approach, the target nucleic acids are first amplified, and thenwith or without additional isolation or purification from theamplification mixture sample, contacted with an AP site probe, anenhancer and an AP endonuclease.

In another approach, the target amplification and target detection aresimultaneously run in the same reaction mixture. This approach allowsthe detection of a target nucleic acid in real time by detectingtail-cleavage products during the amplification reaction. In someembodiments, a fluorescent signal generated by cleavage of a tail with afluorescent dye is visually detected. Simultaneous amplification anddetection also allows measurement of an amount of target nucleic acidsin the test sample. When the target amplification and detection are runsimultaneously, reaction conditions (i.e., salt composition and reactioncomponent concentrations, pH, and temperature of the reaction) aredesigned such that they support both amplification and detection. Also,the detection and amplification processes must not interfere with eachother such that the combined assay is disabled. For example, if PCR isused to amplify the target DNA, the AP endonuclease used should becatalytically active at elevated temperatures (typically 80-100° C.)used in PCR to melt double stranded DNA during the amplification cycles.This can be achieved by use of thermostable AP endonuclease (see, PCTPublication No. WO 93/20191, herein incorporated by reference), additionto the reaction buffer some special component that increasethermostability of the enzyme, for example, trehalose (see, Carninci,P., et al., Thermostabilization and thermoactivation of thermolabileenzymes by trehalose and its application for the synthesis of fulllength cDNA (1998) Proc. Natl. Acad. Sci. USA, 95, 520-524), or acombination of both these approaches.

By contrast, isothermal amplification techniques generally do notrequire temperature changes during the target amplification and can becarried out over a wide range of temperatures, i.e., from 20° C. to 70°C. The selected temperature will depend on the thermal stability of theenzymes used and optimal assay conditions. Isothermal amplificationassays can be combined with known AP endonucleases. Examples of such APendonucleases include without limitation Endonuclease IV from E. colithat is stable up to 70° C., human APE endonuclease, and yeast APendonuclease.

As illustrated in FIG. 6, the invention further provides foramplification of a target sequence using primers with internal APendonuclease cleavage sites having a sequence structure (NA₁-L)_(m)-NA₂,where NA₁ and NA₂ are nucleic acid sequences complementary to the targetnucleic acid, L is an endonuclease-cleavable linker and m is from 0 to100. Primers having internal AP endonuclease cleavage sites hybridizedto a target nucleic acid can function as primers for a polymeraseextension once a 3′ functional tail or an internal linker cleavage site(pL) simulating an abasic site is cleaved, leaving an available 3′-OHgroup. In a primer that contains several pL sites, an AP endonucleasecleaves pL sites, thereby generating 3′-OH priming sites for thepolymerase. The polymerase synthesizes a complementary nucleic acidsequence extending from the newly formed primer that displaces apreviously synthesized complementary nucleic acid strand from adownstream pL cleavage site. Each synthesized strand serves as atemplate for a forward primer. With this amplification scheme, thenumber of target nucleic acid copies that can be generated from oneprimer is equal to the number of pL linker cleavage sites. To facilitateexponential amplification of a desired amplicon, at least one of theprimers should have more than one pL linker while the other primer hasat least one pL linker. Greater numbers of pL linkers within the primersequences will result in more efficient amplification of a desiredtarget sequences.

For amplification of a nucleic acid sequence of interest from an AP siteprimer, it is preferable that polymerase activity in the reactionmixture dominates over endonuclease activity. This could be achieved,for example, by balancing of the relative enzyme concentrations(polymerase vs. endonuclease). Preferably, the endonuclease cleaves pLlinkers only when a primer or product of its extension is duplexed witha target nucleic acid strand. Further, the polymerase used foramplification using an AP site primer preferably lacks the 5′-3′ exo orendonuclease activity and 3′-5′ exonuclease activity (proof reading).Finally, the polymerase used in an AP site primer amplification schemepreferably “reads through” or extends over templates that haveincorporated pL linker sites. It is also preferred that the polymeraseincorporates any natural base against the pL linker during chainelongation. Under appropriate reaction conditions, the activities of thepolymerase and the endonuclease should allow for isothermalamplification of both strands of a desired amplicon within a nucleicacid target sequence located between and including the sequences of aforward and a reverse primer. Nucleic acid amplification using AP siteprimers can be combined with nucleic acid detection resulting fromfunctional cleavage because AP site cleavage in either instance iscatalyzed by the same endonuclease.

B. Detection of Nucleic Acid Polymorphism Using AP Site Tail CleavingTechnology

AP site probes are particularly suited for DNA genotyping or detectionof two related target nucleic acids that share essentially the samesequence and that are different by a number of bases within the sequenceof interest. Most commonly, the difference in the target DNA sequencesof interest are as small as one base (SNP). AP endonucleases generallybind to the DNA on either side from an abasic site and are affected bymismatched base pairs residing in proximity to their preferred enzymebinding site. A mismatched base pair that resides within the region ofan AP endonuclease binding site has a negative effect on theenzyme-DNA-substrate binding, and consequently impedes the catalyticrate of tail-cleavage, as measured by a detectable reporter groupsignal. AP endonucleases identify mismatched base pairs located in theregion of their binding sites by preferentially cleaving the functionaltails R of an AP site probe duplexed with a target nucleic acid sequencehaving matched base pairs located outside the enzyme binding region incomparison to cleaving the tail R of a probe duplexed with a targetnucleic acid having mismatched base pairs in the enzyme binding region.

AP site probes find particular use in detecting base pair mismatchesthat potentially exist at a known or suspected location in a targetnucleic acid. Usually in such assays, two or more different AP siteprobes are contacted with one or more target nucleic acids in a sample,each probe having a nucleic acid sequence differing at one or more basesand distinctly detectable reporter groups. For instance, the two or moreAP site probes could each have a functional tail comprising afluorophore with detectably distinct emission wavelengths, for instance6-fluorescein or Green Dye (FIG. 7, structure 6) and Yakima Yellow (FIG.7, structure 5). When Endonuclease IV from E. coli is used in the assay,discriminatory cleavage of a functional tail R is most pronounced whenthe base pair mismatches are located at the 3′ end of an AP site probein a probe-target nucleic acid duplex. Preferably, the mismatch ispositioned within 8 nucleotides from the 3′ end of the probe, morepreferably at the 7, 6, 5, 4 or 3 position from the 3′ end of the probe,and most preferably at the 1 or 2 position from the 3′ end of the probe,where position 1 is the 3 ′ end nucleotide. In a most preferredembodiment the mismatch is located at position 2 from the 3′ end of theprobe. Base pair mismatch identification assays using an AP site probecan be conveniently carried out in combination with amplificationsystems, particularly with isothermal amplification systems.

An AP site probe used in base pair mismatch identification generally isabout 6-18 nucleotides in length, more preferably about 6-16 nucleotidesin length. If the probe is comprised entirely of naturally occurringbase pairs, it is preferably about 10-16 nucleotides. AP site probesfrom a universal probe library also find use in tail cleavage base pairmismatch identification assays. Universal library oligonucleotides of 5,6, 7 or 8 nucleotides can be used, particularly those which arecomprised at least in part of modified bases.

C. AP Site Probes Constructed from a Universal Library

The present invention contemplates AP site probes constructed from auniversal library. By “universal library” is intended all possiblepermutations of the naturally occurring nucleotide bases for aparticular nucleotide length. Generally, a universal library for anoligonucleotide of n nucleotides is 4^(n) members. For example, theuniversal library for an oligonucleotide of 6 nucleotides in length is4⁶ or 4096 members. In certain embodiments, an AP Endonucleasetail-cleavage assay will use universal oligonucleotide libraries of 6, 7or 8 nucleotides in length. To increase the hybridization meltingtemperatures of some or all members of a universal library, theoligonucleotides can contain incorporated modified bases, such as thosedescribed above.

D. Microfluidics

Methods for target nucleic acid detection and/or amplification using oneor more AP site probes are well suited for large-scale, high-throughput,and parallel processing, particularly when carried out at micro scalevolumes, for instance in capillary-design microfluidics devices.Applicable microfluidic devices and systems are commercially availablefrom, for example, Caliper Technologies (Mountain View, Calif.,www.calipertech.com) and Aclara Biosciences (Mountain View, Calif.,www.aclara.com). Microfluidic devices are applicable for carrying outcombined detection and amplification procedures at micro scale volumes.Microfluidic systems and devices of potential use in carrying out thepresent methods are described, for example, in U.S. Pat. Nos. 6,558,960;6,551,836; 6,547,941; 6,541,274; 6,534,013; 6,558,945; 6,399,952,6,306,273; and 6,007,690, and in U.S. Publication Nos. 2003/0027352,2003/0017467, 2003/17461, 2002/0092767.

The following examples are provided to illustrate, but not to limit, theinvention.

IV. EXAMPLES Example 1

This Example demonstrates the efficacy of an Endonuclease (Class II APendonuclease) tail-cleavage assay.

Assay Design and Oligonucleotide Component Structures (SEQ ID NOS:3-5):

Two probes were used in this example experiment. These probes weredesigned complementary to a target oligonucleotide and they share thesame oligonucleotide structure and a 5′-conjugated quencher (Q) moiety.Structure of the quencher is shown on FIG. 8 (structure #15). Firstprobe was conjugated to a fluorescein dye via a rigid, hydroxyprolinollinker at the 3′-end. Structure of the 3′-tail used (structure #8) isshown on FIG. 7. Second probe contained a flexibleendonuclease-cleavable linker that was created by incorporation of anadditional, propandiol linker (—O—PO₂—O—CH₂CH₂CH₂—O—) between 3′-OHgroup of the first probe and the hydroxyprolinol linker. An enhanceroligonucleotide was used in the assay to support the tail cleavagereaction. The target has one unpaired base between the duplexes of theprobe and enhancer.

The experiment was carried out using an LightCycler™ (Idaho TechnologyInc.). Samples were prepared on ice by mixing concentrated componentstock solutions and then quickly transferred to the instrument chamberwhere they were heated to and kept at 40° C. Final concentration of thereaction components: probe=enhancer=150 nM, target=5 nM, E. coliEndonuclease IV=0.04 Units/μL, Bovine Serum Albumin (BSA)=0.025% in 20mM Tris-HCl (pH8.5), 5 mM MgCl₂. The reaction volume was 10 μL. The timeof the fluorescence recording cycle was 40 sec.

The results are depicted in FIG. 11. When probe with the rigid linker isin a mixture with the target and enhancer oligonucleotides, a strongfluorescence signal was detected over the time of the experiment.Endonuclease IV recognized the target-probe-enhancer complex and cleavedthe fluorescein-liker moiety of the probe, releasing the dye from thequenching effect of the 5′-Q-tail. Absence of the enhancer resulted inreduction of the signal, whereas removal of the target from the systemprovided no fluorescence signal at all (background signal), indicating avery high level of the reaction specificity.

When the 3′-endonuclease-cleavable tail was elongated by incorporationof a propandiol linker, the probe with a flexible 3′-tail, similareffects were observed. In contrast to the first probe, the presence ofthe enhancer was less critical when using the flexible 3′-tail, butfluorescence increase was detected in absence of the target. A high,almost quantitative yield of the target-specific tail-cleavage reactionindicates the cycling mode of the reaction since the probes were takenin 30-fold excess over the target.

Example 2

This Example illustrates that the efficiency of the AP endonucleasetail-cleavage reaction depends on the balance between the hybridizationproperties of the probes and temperature of the reaction. Probescomplementary to target of 11, 9, 7 and 6 nucleic acids in length wereprepared.

Assay Design, Component Structures (SEQ ID NOS:3-5) and MeltingTemperatures (T_(m)):

Q is a 5′-conjugated quencher (structure #15) shown on FIG. 8. FAM is anendonuclease cleavable tail comprising of a fluorescein dye and linkerthat are shown on FIG. 7 (Structure 8). In addition to the shown 6-merprobe, a base-modified 6-mer probe was prepared. All three T bases inthis probe were replaced with 5-hydroxybutynyl uridine that provides aduplex stabilizing effect.

This experiment was done on an ABI PRISM™ 7700 Sequence Detector. Thereaction volume was 10 μL. Samples were prepared on ice and then quicklytransferred to the instrument chamber where they were heated to and keptat 30° C. The time of the fluorescence recording cycle was 30 sec. Finalcomponent concentrations in the samples: probe=enhancer=150 nM, target=5nM, E. coli Endonuclease IV=0.04 Units/μL in 20 mM Tris-HCl (pH8.5), 5mM MgCl₂. The results are depicted in FIG. 12. The tail of the longest11-mer probe (T_(m)=45° C.) was not cleaved in the presence of theenhancer and target at 30° C. whereas fluorescence was detected at 40°C. The elevated stability of the duplex formed with the longest probeinterferes with the target recycling efficiency. The tail of the shorter9-mer probe (T_(m)=35° C.) was efficiently cleaved. Efficiency of thetarget-specific tail-cleavage depends on how the hybridizationproperties of the probes are balanced with the reaction temperature.Generally, the greater the difference between the probe T_(m) andreaction temperature, the lower the fluorescent signal over increasingreaction time. Target-specific cleavage of the tail of the 6-mer probe(T_(m)=2° C.) was not observed. However, when the duplex-stabilizingbases were incorporated into this probe (T_(m)=13° C.), fluorescentsignal was detected.

Example 3

This Example illustrates the substrate specificity of E. coliEndonuclease IV. In this set of experiments (SEQ ID NOS:3-7), theenhancer was positioned along the target sequence to provide a gapbetween the duplexes of the probe and enhancer of 0, 1 or 2 nucleotides.

Q is a 5′-conjugated quencher (structure #15) shown on FIG. 8. FAM is anendonuclease cleavable tail comprising of a fluorescein dye and linkerthat are shown on FIG. 7 (Structure 8).

The experiment was done on a Rotor-Gene 3000 (Corbett Research, Sydney,Australia). The reaction volume was 10 μL. Samples were prepared on iceand then quickly transferred to the Rotor-Gene chamber where they wereheated to and kept at 40° C. The time of the fluorescence recordingcycle was 40 sec. Final component concentrations in the samples:probe=enhancer=150 nM, target=5 nM, E. coli Endonuclease IV=0.04Units/μL in 20 mM Tris-HCl (pH8.5), 5 mM MgCl₂.

The results are depicted in FIG. 13. The greatest fluorescent signal wasobserved when a gap of 1 nucleotide was present between the probe andthe enhancer hybridized to the target. A probe-target nucleicacid-enhancer complex with no unpaired bases between the probe andenhancer showed little detectable fluorescent signal, presumably due togreatly diminished cleavage of the fluorescent tail. Complexes having atwo base gap performed much better than complexes without a base gap.Complexes having a one base gap were the preferred substrate for theEndonuclease IV, most likely because this complex most closely resemblesa natural substrate for the enzyme.

Example 4

This Example illustrates the application of the tail cleaving assay tothe discrimination of single base pair mismatch. Endonuclease IVdiscriminates single base-pair mismatches, particularly those located atthe 3′-end of AP site probe hybridized with a target nucleic acid.

Assay Design and Oligonucleotide Component Structures (SEQ ID NOS:8-11):

41-mer target

Q is a 5′-conjugated quencher (structure #15) shown on FIG. 8. FAM is anendonuclease cleavable tail comprising of a fluorescein dye and linkerthat are shown on FIG. 7 (Structure 8). The probes used in this exampleare a 14-mer (T_(m)=60° C.) and 10-mer (T_(m)=48° C.) oligonucleotides.The enhancer does not need to cycle in the reaction and it has anelevated T_(m) of 70° C. In addition to the target sequence shown above,eighteen 41-mer target nucleic acid sequences were synthesized to studythe Endonuclease IV tail-cleavage activity in the presence of mismatchedprobe/target complexes. These DNA targets differed from the fullymatched sequence by one base such that they formed three single basemismatches with every probe nucleotide located within six bases closestto the 3′-end. One target hybridized with the probes provided a G/Tmismatch located at the position eight from the 3′-end of the probes.Variable bases within the target sequence are underlined. The reactionswere run under standard conditions that are described in the Examples2-4. The initial rate of tail cleavage was measured for every targetnucleic acid/probe combination as a function of the reactiontemperature. The data for the 14-mer and 10-mer probes are shown in FIG.14 and FIG. 15, respectively.

Excellent mismatch discrimination is observed when the mismatch isplaced 1 or 2 bases from the 3′-end. Probes shorter than 14-mers maydiscriminate mismatches more effectively. In other experiments (FIG.15), a 10-mer probe showed a 4-fold slower rate of the tail cleavage atthe optimal reaction temperature, as measured by fluorescence units perminute, in comparison to a 14-mer probe. However, a greater overallrange in detectable signal between matched and mismatched duplexes wasobserved when using a 10-mer probe. Because of the greater thermodynamiccontribution of each nucleotide base pair in shorter probes relative tooverall duplex energy, shorter probes appear to more effectivelydiscriminate between complementary and mismatched probe-target duplexes.

Both thermodynamic and enzyme efficiency contribute to SNPdiscrimination in an AP endonuclease tail cleaving assay. With regard tothermodynamics, at a given temperature, probes bind with differentefficiencies to the matched and mismatched sites. With regard to enzymeefficiency, the endonuclease cleavage efficiency is decreased when thebase pair mismatch is located close to the 3′-end of the probe. Thefurther the mismatch from the 3′-end of the probe, the more diminishedeffect it had on enzyme tail-cleavage efficiency. Optimal mismatchdiscrimination was achieved in cases when mismatches were located at thevery 3′-end of the probe (position 1) or at the next base pair (position2). When mismatches are at position 2, fluorescent signal is essentiallyundetectable.

Stable mismatches like T/G were not as effectively discriminated. Incontrast, A/C, T/C, C/C mismatches were discriminated very well.Unexpectedly, a relatively unstable T/T-mismatch at positions 4 and 5allowed for detectable probe tail-cleavage although the maximum of theprobe tail-cleavage occurred at lower temperatures.

Example 5

This Example illustrates the application of the tail cleaving assay fora post-PCR detection of a single nucleotide polymorphism in humangenomic DNA using two AP site probes from a universal library 8nucleotides in length.

Assay Design and Oligonucleotide Component Structures:

                        -T- (polymorphism) 3′............CTACCTAACTA CACAGGCACAGAGAAA.........5′                            TCCGTGTC-3′  Enhancer (T_(m) = 39° C.)              5′-Q-attGat G T-FAM-3′       First Probe (T_(m) = 32° C.)              5′-Q-attGat a t-YD-3′        Second Probe (T_(m) = 32° C.)

A fragment of the target sequence around the polymorphism is shownabove. (SEQ ID NO:12). The T/C mismatch is underlined. First and secondprobe were labeled with a fluorescent tails that are shown on FIG. 7,structure #8 (FAM) and #7 (YD) respectively. Q is a 5′-conjugatedquencher (structure #15) shown on FIG. 8. The A and T bases aresubstituted with modified bases “a” and “t”.

Three individual samples of the human genomic DNA that were priorgenotyped as T-homozygous, T/C-heterozygous and C-homozygous at thepolymorphism of interest were amplified in an asymmetric PCR. PCR wereperformed on ABI PRISM™ 7700 Sequence Detector using forwardCAAACTTTGTCCTTGGTCTA (SEQ ID NO:13) and reverse TTCTTTTACCACTCCCCCTT(SEQ ID NO:14) primers and a PCR cycling profile: 2 min 50°-2 min 95°-(5sec 95°-20 sec 56°-30 sec 76°)×50 times.

PCR Reaction Composition and Concentration:

Forward primer—2 μM; reverse primer—100 nM; target DNA—1 mg/μl;JumpStart Taq DNA polymerase—0.08 U/μl; Uracil-N-Glycosylase—0.01 U/μl;dATP, dCTP and dGTP—125 μM; dUTP—250 μM in 40 mM NaCl, 20 mM Tris-HCl(pH8.7), 2.5 mM MgCl₂. PCR reaction volume was 50 μl.

After 50 cycles, 5 μl of each PCR reaction was mixed with 5 μl of asolution that contained both AP site probes and the enhancer atconcentration 300 nM and E. coli Endonuclease IV—0.08 U/μl in 40 mMTris-HCl (pH8.5), 10 mM MgCl₂. Reaction mixture were transferred to theABI PRISM™ 7700 Sequence Detector chamber where they were heated to andkept at 30° C. Fluorescence was detected in FAM and VIC channels of theinstrument. Results are shown on FIG. 16. Cleavage of the AP site probesis in agreement with the DNA allelic composition. Only first probe wascleaved in case of C-homozygous DNA and the increase of the fluorescentsignal over time was detected in the FAM channel respectively. Thesituation is reversed when T-homozygous DNA was used whereas both probewere cleaved in the reaction mixture containing the heterozygous DNAamplified.

Example 6

This Example illustrates that cleavage of a functional tail R from an APsite probe does not effect on the probe hybridization properties. Twosamples were prepared by mixing a complementary targetoligodeoxyribonucleotide 5′-CAAGGACCGAGTC-3′ (SEQ ID NO:15) in 5 mMMgCl₂, 20 mM Tris-HCl (pH8.5) with ODN probes 5′-Q-ACTCGGTCCTT-FAM-3′(SEQ ID NO:16) and 5′-Q-ACTCGGTCCTT-3′(SEQ ID NO:17) respectively. Q isa 5′-conjugated quencher (structure #15) shown on FIG. 8. FAM is anendonuclease cleavable tail comprising of a fluorescein dye and linkerthat are shown on FIG. 7 (Structure 8). Denaturation profiles of theduplexes are shown on FIG. 17. These profiles were obtained bymonitoring the sample absorbance (A₂₆₀) vs. temperature (0.4° C./min).The target ODN was taken in 1.2 fold excess over the probes that were at1 μM concentration.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application. Allpublications, patents, and patent applications cited herein are herebyincorporated herein by reference in their entirety for all purposes.

1. A method of detecting a target nucleic acid in a sample, comprising:a) contacting the sample with at least one AP site probe and an APendonuclease, under conditions sufficient to allow the AP site probe tohybridize to the target nucleic acid and form a reaction mixture,wherein said AP site probe comprises an oligonucleotide NA thathybridizes to the target nucleic acid and a functional tail R comprisinga detectable reporter group, said functional tail R attached via aphosphodiester bond of a phosphate group to the 3′ terminal nucleotideof the NA, wherein the reporter group is not detected when thefunctional tail R is attached to the NA; and b) incubating the reactionmixture under reaction conditions sufficient to allow said APendonuclease to cleave the phosphodiester bond attaching the functionaltail R to the 3′ terminal of the NA, wherein the AP endonucleasepreferentially cleaves the phosphodiester bond attaching the tail R tothe NA when the NA is hybridized with a complementary target nucleicacid sequence in comparison to when the NA is unhybridized or hybridizedto a non-complementary target nucleic acid; and c) detecting thereporter group on the cleaved functional tail R, whereby the targetnucleic acid is detected.
 2. The method of claim 1, further comprisingcontacting the sample with an enhancer oligonucleotide, wherein the5′-end of said enhancer oligonucleotide hybridizes to the target nucleicacid on the 3′ side of the hybridized AP site probe, wherein a gap of 1,2 or 5 unpaired bases resides between the enhancer oligonucleotide andthe AP site probe hybridization locations with the target nucleic acid.3. The method of claim 2, wherein said AP site probe is covalentlylinked to the 3′ end said enhancer.
 4. The method of claim 1, furthercomprising a quencher molecule attached to the 5′ end of the NA of saidAP site probe via a non-cleavable linker.
 5. The method of claim 1,wherein the cleavage of the phosphodiester bond results in a hybridizedNA having a free 3′-OH.
 6. The method of claim 5, further comprisingcontacting the sample with a nucleic acid polymerase, and furthercomprising amplifying the target nucleic acid, said amplifyingcomprising incubating the sample under reaction conditions sufficient toallow the polymerase to extend the hybridized NA in a template-specificmanner.
 7. The method of claim 6, wherein said amplifying is isothermalamplification.
 8. The method of claim 5, wherein the sample is incubatedunder reaction conditions that simultaneously allow the AP endonucleaseto cleave the phosphodiester bond of the AP site probe and thepolymerase to extend the cleaved AP site probe in a template-specificmanner.
 9. The method of claim 1, wherein the NA of said AP site probeis 3-200 nucleotides in length.
 10. The method of claim 1, wherein thefunctional tail R is attached to the phosphate group through ahydroxyprolinol linker.
 11. The method of claim 1, wherein the reportergroup is a fluorophore.
 12. The method of claim 1, wherein the APendonuclease is a Class II AP endonuclease.
 13. The method of claim 12,wherein the Class II AP endonuclease is an E.coli Endonuclease IV. 14.The method of claim 1, wherein the target nucleic acid is attached to asolid support.
 15. The method of claim 1, wherein the AP site probe isattached to a solid support.
 16. The method of claim 2, wherein theenhancer is attached to a solid support.
 17. The method of claim 1,wherein step (a) further comprises contacting a second AP site probewith the target nucleic acid, wherein said first probe comprises a NAportion comprising at least one base difference from the NA portion ofsaid second probe, and wherein said first probe comprises a reportergroup that is distinguishably detectable from the reporter group of saidsecond probe.
 18. The method of claim 17, wherein the reporter group ofsaid first probe and said second probe comprises a fluorophore, andwherein the fluorophore of said first probe comprises a distinguishablydetectable emission wavelength from the fluorophore of said secondprobe.
 19. The method of claim 17, wherein said at least one basedifference between the NA of said first probe and the NA of said secondprobe comprises a base difference at position 1, 2, 3 or 4 from the 3′end of said probes.
 20. The method of claim 17, wherein said at leastone base difference between the NA of said first probe and the NA ofsaid second probe comprises a base difference at position 1 or 2 fromthe 3′ end of said probes.
 21. The method of claim 1, wherein said atleast one AP site probe comprises a plurality of AP site probes, whereinthe NA portion of said probes are members of a universal library. 22.The method of claim 21, wherein the NA portion of said AP site probemembers is 5-8 nucleotides in length.
 23. The method of claim 9, whereinsaid AP site probe members further comprise at least one modified base.24. A method of detecting a target nucleic acid in a sample, comprising:a) contacting the sample with at least one AP site probe and an APendonuclease, under conditions sufficient to allow the AP site probe tohybridize to the target nucleic acid and form a reaction mixture,wherein said AP site probe comprises an oligonucleotide NA thathybridizes to the target nucleic acid, a functional tail R comprising aquencher molecule, said functional tail R attached via a phosphodiesterbond of a phosphate group to the 3′ terminal of the NA, and a reportergroup attached via a non-cleavable linker to the 5′ terminal of the NA,wherein the reporter group is not detected when the functional tail R isattached to the NA; and b) incubating the reaction mixture underreaction conditions sufficient to allow said AP endonuclease to cleavethe phosphodiester bond attaching the functional tail R to the 3′terminal of the NA, wherein the AP endonuclease preferentially cleavesthe phosphodiester bond attaching the tail R to the NA when the NA ishybridized with a complementary target nucleic acid sequence incomparison to when the NA is unhybridized or hybridized to anon-complementary target nucleic acid; and c) detecting the reportergroup upon cleavage of the functional tail R, whereby the target nucleicacid is detected.
 25. The method of claim 1, wherein said target nucleicacid is a product of an amplification reaction.
 26. The method of claim25, wherein said amplification reaction is polymerase chain reaction.27. The method of claim 25, wherein said amplification reaction ispolymerase chain reaction and said method uses thermostableendonuclease.
 28. The method of claim 25, wherein said amplificationreaction is an isothermal amplification reaction.